Isolation and characterization of Pseudomonas aeruginosa bacteriophage phikF77 mutants]

It was found that phage phi kF77 is resistant to all known Pseudomonas aeruginosa restriction systems. Three types of mutants (dc-) which were unable to grow on different restrictive strains were isolated. All of them belong to one complementation group. Some of these mutations affected also the number of nicks in phage phi kF77 DNA and increased phage resistance to temperature treatment. It may be supposed that genes responsible for antirestriction mechanisms and introduction of nicks into DNA are connected in definite way.     

DNA nicks inflicted by restriction endonucleases are repaired by a RecA- and RecB-dependent pathway in Escherichia coli

Two mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at EcoRI sites in vivo. Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli. In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at non-cognate EcoRV sites in the bacterial genome. EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.     

Structure of discontinuities in kinetoplast DNA-associated minicircles during S phase in Crithidia fasciculata

Kinetoplast DNA (kDNA) is a novel form of mitochondrial DNA consisting of thousands of interlocked minicircles and 20–30 maxicircles. The minicircles replicate free of the kDNA network but nicks and gaps in the newly synthesized strands remain at the time of reattachment to the kDNA network. We show here that the steady-state population of replicated, network-associated minicircles only becomes repaired to the point of having nicks with a 3′OH and 5′deoxyribonucleoside monophosphate during S phase. These nicks represent the origin/terminus of the strand and occur within the replication origins (oriA and oriB) located 180° apart on the minicircle. Minicircles containing a new L strand have a single nick within either oriA or oriB but not in both origins in the same molecule. The discontinuously synthesized H strand contains single nicks within both oriA and oriB in the same molecule implying that discontinuities between the H-strand Okazaki fragments become repaired except for the fragments initiated within the two origins. Nicks in L and H strands at the origins persist throughout S phase and only become ligated as a prelude to network division. The failure to ligate these nicks until just prior to network division is not due to inappropriate termini for ligation.     

T98G glioma cells have nicks in DNA in quiescent phase

Human glioblastoma-derived cell line, T98G, is arrested in the G1 phase of the cell cycle when serum is deprived. Using this cell line, we investigated the relation between the cell cycle and DNA single-stranded breaks, "nicks," by an in situ nick-translation method. When T98G cells were cultured without serum for 60 h, many small cells with condensed chromatin and scanty cytoplasm appeared. These small cells that were immunohistochemically considered to be in the G0 or early G1 phase had many nicks in DNA. When serum was added, these small cells with nicks disappeared within 1 to 4 h. VP-16, a DNA topoisomerase II inhibitor, delayed the disappearance of these small cells with nicks. This indicated that the action of DNA topoisomerase II on the chromatin is required to repair nicks in T98G glioma cells and to promote the progression from the quiescent to the proliferating phase.     

The effect of histamine on the growth of cultured fibroblasts isolated from normal and keloid tissue

Cultured fibroblasts derived from human keloid tissue are presented as a possible model system for studying the genetic regulation of cell growth. Histamine is shown to have a marked effect on the growth of cultured fibroblasts. A small increase in growth rate is seen during the log phase of the culture cycle and a 50% increase in cell number is observed during the plateau phase. Differences in the extent of growth stimulation are observed between strains isolated from different individuals. While most strains showed approximately 50% stimulation, a few were not stimulated and some strains gave a 100% or greater increase in cell number due to histamine. This phenotypic difference in extent of growth stimulation in response to histamine cannot be attributed to the gene or genes for keloid formation. However, elevated levels of histamine in vivo may be a contributing factor to the abnormal cell growth observed in this disorder. The extent of growth stimulation due to histamine decreases with repeated subculturing.     

rDNA enhancer affects replication initiation and mitotic recombination: Fob1 mediates nucleolytic processing independently of replication

To investigate the influence of the ribosomal DNA enhancer on initiation of replication and recombination at the ribosomal array, we used yeast S. cerevisiae strains with adjacent, tagged rRNA genes. We found that the enhancer is an absolute requirement for replication fork barrier function, while it only modulates initiation of replication. Moreover, the formation of monomeric extrachromosomal ribosomal circles depends on this element. Our data indicate that DNA double-strand breaks occur at specific sites in the parental leading arm of replication forks stalled at the replication fork barrier. Additionally, nicks upstream of the replication fork barrier were visualized by nucleotide-resolution mapping. They coincide with essential sequences of the mitotic hyperrecombination site HOT1, which previously has been determined at ectopic sites. Interestingly, these nicks are strictly dependent on the replication fork blocking-protein (Fob1), but are replication independent, suggesting that intrachromosomal ribosomal DNA recombination may occur outside of S phase.     

A disruption of pachytene DNA metabolism in male mice with chromosomally-derived sterility

DNA metabolism was analyzed in spermatocytes of mice that were sterile either because of X-autosome or autosome-autosome translocations, or because of trisomy. In the strains analyzed, spermatogenic development is arrested by metaphase I or soon thereafter. In all such strains, a disruption of the normal pattern of pachytene DNA metabolism occurred. Prepachytene metabolism appeared normal. Disruption was manifest in both the level of endogenously generated nicks during pachytene and in the distribution of nicks among the different DNA sequence classes. Nicking was more intense in the steriles and tended to be randomized in distribution. Satellite DNA underwent pachytene nick-repair in the steriles but not in fertile controls. The repair capacity of spermatocytes from steriles was equal to that of the fertiles; the higher frequency of nicks in the steriles was due to a persistence of nicking activity.     

Biological role of DNA methylation: sequence-specific single-strand breaks associated with hypomethylation of GATC sites in Escherichia coli DNA.

The effect of methylation of GATC sites in Escherichia coli DNA on the formation of single-strand breaks was studied with dam+, dam mutant, and Dam-overproducer strains. Single-strand breaks have been observed in dam mutant cells predominantly at TpT and, to a lesser extent, at CpC. In dam mutant cells harboring pTP166 (a plasmid containing the dam gene), no such nicks were observed.     

Adherence as a method of differentiating virulent and avirulent strains of Vibrio parahaemolyticus.

Usually only Kanagawa-positive strains of Vibrio parahaemolyticus are considered virulent; yet, a significant portion of V. parahaemolyticus food poisonings appear to be caused by Kanagawa-negative strains. Therefore, additional and more accurate measurements of a strain's food-poisoning potential are needed. Adherence of V. parahaemolyticus to human fetal intestinal (HFI) cells in vitro seems to offer this information. All strains of V. parahaemolyticus adhered to the HFI cells, but the degree of adherence was related to a number of factors. These included the age of the culture, the strain's Kanagawa reaction and source, the length of time the bacteria were exposed to the HFI cells, and the composition of the growth medium. Cells harvested during the late log phase of growth adhered more intensely than those harvested from the late stationary phase. Virulent strains, i.e., those involved in food poisoning, were observed to have a high adherence ability regardless of their Kanagawa reaction, whereas Kanagawa-negative strains isolated from seafood exhibited weak adherence intensities. Kanagawa-positive strains isolated from seafood adhered strongly to the HFI cells. The difference between the virulent and avirulent strains was quantitative in nature, and the greatest differences in adherence intensities were observed after short (10 to 15 min) exposure times. The presence of ferric iron in the growth medium was found to increase the adherence intensities of the virulent strains.     

Psoralen cross-linking as probe of torsional tension and topological domain size in vivo

DNA within a cell is organized with unrestrained torsional tension, and each molecule is divided into multiple individual topological domains. Psoralen photobinding can be used as an assay for supercoiling and topological domain size in living cells. Psoralen photobinds to DNA at a rate nearly linearly proportional to superhelical density. Comparison of the rate of photobinding to supercoiled and relaxed DNA in cells provides a measure of superhelical density. For this, in vivo superhelical tension is relaxed by the introduction of nicks by either ionizing radiation or photolysis of bromodeoxyuridine in the DNA. Since nicks are introduced in a random fashion, the distribution of nicks is described by a Poisson distribution. Thus, after nicking, the fraction of topological domains containing no nicks is described by the zero term of the Poisson distribution. From measurement of the number of nicks introduced in the DNA and the fraction of torsional tension remaining, an average topological domain size can be estimated. Using this logic, procedures were designed and described for measuring supercoiling and domain size at specific sites in eukaryotic genomes.     

The NS1 protein of the autonomous parvovirus minute virus of mice blocks cellular DNA replication: a consequence of lesions to the chromatin?

The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice interferes with cell division and can cause cell death, depending on the cell transformation state. Upon infection, the synthesis of NS1 protein is massively initiated during S phase. In this article, we show that minute virus of mice-infected cells accumulate in this phase. To investigate the link between NS1 accumulation and S-phase arrest, we have used stably transfected cells in which NS1 expression is under the control of a glucocorticoid-inducible promoter (the long terminal repeat of mouse mammary tumor virus). NS1 expression interferes with cell DNA replication, and consequently, the cell cycle stops in S phase. NS1 expression also induces nicks in the cell chromatin, as detected by an in situ nick translation assay. The nicks are observed several hours before any cell cycle perturbation. As cell cycle arrest is a common consequence of DNA damage, we propose that NS1 exerts its cytostatic activity by inducing lesions in cell chromatin.     

In vitro amino acid requirements of Bacillus anthracis cells

In the process of batch cultivation in a synthetic medium B. anthracis cells actively consume free amino acids. The maximum amino acid consumption per unit of biomass occurs at the exponential phase of growth, but the consumption of serine is maintained at a high level also during the stationary phase. At the same time the consumption of proline by both vaccine and virulent strains is insignificant at the stationary phase of growth. In contrast to B. anthracis virulent strains, vaccine strains have been shown to consume no lysine, histidine, arginine and tryptophan.     

Accumulation of single strand interruptions within the yeast 2 microns DNA plasmid during replication in a DNA ligase mutant

Summary We have investigated the fate of the yeast 2 m DNA plasmid in strains with a temperature sensitive mutation of DNA ligase. At the restrictive temperature the plasmid DNA collects as an open circular form with single strand interruptions. Both alpha factor pheromone, which arrests cells before the start of S phase, and hydroxyurea, which blocks progression through S phase, prevent the appearance of the open circular form. Thus, interrupted plasmid DNA does not accumulate in the absence of DNA replication. On average the interrupted molecules contain four to five interruptions per newly replicated strand. Most of the interruptions are nicks (breaks in a single phosphate ester bond) rather than gaps (absence of one or more nucleotides in a strand) as judged by the in vitro conversion of the interrupted molecules into a covalently closed form by DNA ligase. Mapping of the position of the interruptions reveals no predominate sites.     

Detection and analysis of extra- and intracellular deoxyribonuclease activities in Lactobacillus plantarum

Extracellular endodeoxyribonuclease activities have been detected in culture supernatant fluids of several Lactobacillus plantarum strains. In the case of Lact. plantarum HER 1325 at least, the extracellular activity is accompanied by a cytoplasmic restriction endonuclease. Among the secreted nucleases, the greatest activity was from Lact. plantarum ATCC 10241. This strain secretes an enzyme, with a molecular mass in the range 10–40 kDa, that cuts double-stranded DNA in a sequence-independent way by initially introducing single-strand nicks in supercoiled molecules, followed by linearization and complete degradation of the substrate.     

The spatio-temporal distribution of single-stranded breaks in nuclear DNA in sections of clawed toad embryos during gastrulation and neurulation

Spatial and temporal pattern and quantities of nicks in nuclear DNA during gastrulation and neurulation was studied using nick-translation in sections of Xenopus laevis embryos. Specific changes in the number of nicks in different mesoderm and ectoderm regions were detected during embryogenesis. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and inner ectoderm layer of early and middle gastrula. The gradient was inverted during transition from gastrula to neurula. At the same time dorso-ventral (in mesoderm) and ventro-dorsal (in outer ectoderm layer) gradients of nuclear labelling were increased. The intensity of nuclear labelling in all parts of embryo as a whole was remarkably higher during neurulation as compared with gastrulation. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and ectoderm during neurulation. A connection between the nicks and differentiation status of the cells during early embryogenesis in amphibians is suggested.     

Nucleotide sequence at the site of single-strand DNA breaks in Pseudomonas aeruginosa bacteriophage phi kF77

It is known that DNA molecules from the phage group phi k specific to Pseudomonas aeruginosa possess single-strand breaks (nicks). The sequences around the nicks in the bacteriophage phi kF77 DNA have been determined by various methods. In addition, an EcoRV-HindIII fragment, containing a nick, was cloned into the plasmid pUC9 and sequenced by Maxam-Gilbert technique. The sequence common for all nicks was CCTAohpCTCCGG.     

Adherence of Listeria monocytogenes strains to stainless steel coupons

An assay was developed to measure the number of Listeria monocytogenes cells adhering to stainless steel, and was used to investigate the adherence of 111 strains of the organism, which included representatives with respect to serotype, carriage of plasmids, source and persistence in the food processing environment. Growth and adherence curves of four L. monocytogenes strains over 48 h were obtained. While the growth curves of all four micro-organisms were seen to reach similar levels at stationary phase, there was still substantial variation among the adherence curves. In addition, a scatter-graph of growth vs adherence counts at 24 h showed poor correlation. These factors indicated that interstrain variation in adherence at stationary phase is due to factor(s) intrinsic to each strain of L. monocytogenes. Persistent strains were found to adhere in significantly greater numbers than sporadic strains, and variation was also found among serotypes, with serotype 1/2c showing significantly greater adherence than serotypes 1/2a and 4b; 4b strains were significantly higher than those of 1/2a strains. No significant difference was found between strains according to source or plasmid carriage.     

Persistence of high-replicative capacity in cultured fibroblasts from nonagenarians

Earlier studies on human fibroblast cultures have demonstrated an inverse relationship between the total number of population doublings (PDs) and donor age. As more recent studies were unable to replicate these findings, we set out to analyze growth characteristics of fibroblast cultures from nonagenarians who represent the extreme of human lifespan. Therefore, we obtained skin biopsies from 68 participants of the Leiden 85-plus Study, all aged 90 years. None of the 68 strains failed to proliferate and all were easily cultured under highly standardized conditions. Within a time window of 30 months, all strains displayed a high and reproducible replicative capacity that was maintained for at least 50 PDs. A decline in mitotic activity was observed between 26 and 81 PDs. Out of the 68 cell strains, 58 strains reached the post-mitotic phase with an onset between 51 and 108 PDs. The growth pattern of each senescent strain was fitted by a piecewise linear model, which allowed calculation of the transition towards the phase of decreased growth speed, as well as by a nonlinear continuous model; goodness-of-fit was high and not different between the models (both > 0.99). Growth characteristics were not associated with morbidity or mortality of the donors. We conclude that fibroblasts from nonagenarians maintain a high-replicative capacity despite a huge variability in the onset of senescence. These results cast further doubt on the association between in vitro growth characteristics of fibroblast cultures and the length of human lifespan.